The cytochromes P450 are a large family of hemoprotein enzymes capable of metabolizing xenobiotics such as drugs, carcinogens and environmental pollutants as well as endobiotics such as steroids, fatty acids and prostaglandins. Some members of the cytochrome P450 family are inducible in both animals and cultured cells, while other forms are constitutive. This group of enzymes has both harmful and beneficial activities. Metabolic conversion of xenobiotics to toxic, mutagenic and carcinogenic forms is a harmful activity. Detoxification of some drugs and other xenobiotic substances is a beneficial activity (Gelboin, Physiol. Rev. 60:1107-1). A further beneficial activity is the metabolic processing of some drugs to activated forms that have pharmacological activity.
Genetic polymorphisms of P450 enzymes result in phenotypically-distinct subpopulations that differ in their ability to perform particular drug biotransformation reactions. These phenotypic distinctions have important implications for selection of drugs. For example, a drug that is safe when administered to most human may cause intolerable side-effects in an individual suffering from a defect in a P450 enzyme required for detoxification of the drug. Alternatively, a drug that is effective in most humans may be ineffective in a particular subpopulation because of lack of a P450 enzyme required for conversion of the drug to a metabolically active form. Accordingly, it is important for both drug development and clinical use to screen drugs to determine which P450 enzymes are required for activation and/or detoxification of the drug. It is also important to identify individuals who are deficient in a particular P450 enzyme.
A cytochrome P450 polymorphism of particular concern results in reduced levels of S-mephenytoin 4'-hydroxylase activity in certain subpopulations. (Kupfer et al., Eur. J. Clin. Pharmacol. 26:753-759 (1984); Wedlund et al., Clin. Pharmacol. Ther. 36:773-780 (1984). Two phenotypes, extensive and poor metabolizers, are present in the human population. Poor metabolizers are detected at low frequencies in Caucasians (2-5%) but at higher frequencies in the Oriental population (.about.20%) (Nakamura et al., Clin. Pharmacol. Ther. 38:402-408 (1985); Jurima et al., Br. J. Clin. Pharmacol. 19:483-487 (1985) and blacks (.about.12%). 4'-hydroxylation of S-mephenytoin is 3-10 fold higher than that of the R- enantiomer in extensive metabolizers, but the ratio is approximately 1 or less in poor metabolizers (Yasumori et al., Mol. Pharmacol. 35:443-449 (1990). Rates of S-mephenytoin 4'-hydroxylation in liver microsomes are also much higher than those of R-mephenytoin in extensive metabolizers.
There is some evidence that S-mephenytoin 4' hydroxylase activity resides in the cytochrome P450 2C family of enzymes. A number of 2C human variants (designated 2C8, 2C9 and 2C10) have been partially purified, and/or cloned. See Shimada et al., J. Biol. Chem. 261:909-921 (1986); Kawano et al., J. Biochem. (Tokyo) 102:493-501 (1987); Gut et al., Biochem. Biophys, Acta 884:435-447 (1986); Beaune et al., Biochem Biophys. Acta 840:364-370 (1985); Ged et al., Biochemistry 27:6929-6940 (1988)); Umbenhauer et al., Biochemistry 26, 1094-1099 (1987); Kimura et al., Nucleic Acids Res. 15:10053-10054 (1987); Shephard et al., Ann. Humn. Gentc. 53:23-31 (1989); Yasumori et al., J. Biochem. 102:1075-1082 (1987); Relling et al., J. Pharmacol. Ther. 252:442-447. A comparison of the P450 2C cDNAs and their predicted amino acid sequences shows that about 70% of the amino acids are absolutely conserved among the human P450 2C subfamily. Some regions of human P450 2C protein sequences have particularly highly conservation, and these regions may participate in common P450 functions. Other regions show greater sequence divergence regions and are likely responsible for different substrate specificities between 2C members.
There has been considerable controversy as to whether any of the known 2C members encodes the principal human determinant of S-mephenytoin 4' hydroxylase activity, in which the polymorphism discussed above presumably resides. The multiplicity and common properties of cytochromes P450 make it difficult to separate their different forms, especially the minor forms. Even in situations where P450 cytochromes have been isolated in purified form by conventional enzyme purification procedures, they have been removed from the natural biological membrane association and therefore require the addition of NADPH-cytochrome P450 reductase and other cell fractions for enzymatic activity.
The known members of the cytochrome P450 2C family exhibit only low-levels of S-mephenytoin 4'-hydroxylase activity, if any. Moreover, such low levels of activity are not specific for the S-enantiomer. For example f when the cDNA isolated by Kimura et al. (1987), supra, was expressed in HepG2 cells, it metabolized racemic and (R)-mephenytoin but had no (S)-mephenytoin hydroxylase activity, suggesting that the polymorphism in the metabolism of (S)-mephenytoin resides in a different member of the P450 family. As a further example, Yasumori et al. (1991), supra, reported that an allelic variant of 2C9 (Arg.sup.144 Tyr.sup.358 Iso.sup.359 Gly.sup.417) showed a low-level of catalytic activity toward S-mephenytoin in a cDNA-directed yeast expression. However, Srivastava et al., Mol. Pharmacol. 40:69-69 (1991) expressed an identical cDNA in yeast and a Arg.sup.144 Cys.sup.358 Iso.sup.359 Asp.sup.417 variant (2C10 by present nomenclature) but were unable to demonstrate catalytic activity of 2C9 or 2C10 toward S-mephenytoin. Relling et al., J. Pharmacol. Exper. Ther. 252:442-447 (1990), were also unable to demonstrate catalytic activity of an allelic variant of Cys.sup.144 Tyr.sup.358 Ile.sup.359 Gly.sup.417 -2C9 toward S-mephenytoin using a retroviral cDNA expression system in HepG2 cells. In contrast, all of these 2C9 variants metabolized tolbutamide in the various expression systems confirming that failure to observe S-mephenytoin 4'-hydroxylase activity was not due to deficiencies in the expression system.
Based on the foregoing, it is apparent that a need exists to identify and isolate the P450 2C family member representing the principal determinant of S-mephenytoin 4'-hydroxylase activity in humans. There is also a need for stable cell lines expressing the S-mephenytoin 4'-hydroxylase activity. A need is also apparent for methods of screening drugs for safety and efficacy in individuals deficient in S-mephenytoin 4'-hydroxylase activity. There is also a need for methods for diagnosing individuals deficient in S-mephenytoin 4'-hydroxylase activity. The present invention fulfills these and other needs.